Venkataraman Krithika, Zafar Hina, Karzai A Wali
Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, NY 11794, USA.
Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, NY 11794, USA
Nucleic Acids Res. 2014;42(17):11192-202. doi: 10.1093/nar/gku802. Epub 2014 Sep 8.
trans-Translation, orchestrated by SmpB and tmRNA, is the principal eubacterial pathway for resolving stalled translation complexes. RNase R, the leading nonstop mRNA surveillance factor, is recruited to stalled ribosomes in a trans-translation dependent process. To elucidate the contributions of SmpB and tmRNA to RNase R recruitment, we evaluated Escherichia coli-Francisella tularensis chimeric variants of tmRNA and SmpB. This evaluation showed that while the hybrid tmRNA supported nascent polypeptide tagging and ribosome rescue, it suffered defects in facilitating RNase R recruitment to stalled ribosomes. To gain further insights, we used established tmRNA and SmpB variants that impact distinct stages of the trans-translation process. Analysis of select tmRNA variants revealed that the sequence composition and positioning of the ultimate and penultimate codons of the tmRNA ORF play a crucial role in recruiting RNase R to rescued ribosomes. Evaluation of defined SmpB C-terminal tail variants highlighted the importance of establishing the tmRNA reading frame, and provided valuable clues into the timing of RNase R recruitment to rescued ribosomes. Taken together, these studies demonstrate that productive RNase R-ribosomes engagement requires active trans-translation, and suggest that RNase R captures the emerging nonstop mRNA at an early stage after establishment of the tmRNA ORF as the surrogate mRNA template.
由SmpB和tmRNA精心编排的反式翻译是真细菌解决停滞翻译复合物的主要途径。RNase R是主要的无义mRNA监测因子，在一个依赖反式翻译的过程中被招募到停滞的核糖体上。为了阐明SmpB和tmRNA对RNase R招募的作用，我们评估了tmRNA和SmpB的大肠杆菌-土拉弗朗西斯菌嵌合变体。该评估表明，虽然杂交tmRNA支持新生多肽标记和核糖体拯救，但在促进RNase R招募到停滞核糖体方面存在缺陷。为了获得更深入的见解，我们使用了影响反式翻译过程不同阶段的已建立的tmRNA和SmpB变体。对选定tmRNA变体的分析表明，tmRNA开放阅读框的最后一个和倒数第二个密码子的序列组成和定位在将RNase R招募到拯救的核糖体中起着关键作用。对确定的SmpB C末端尾巴变体的评估突出了建立tmRNA阅读框的重要性，并为RNase R招募到拯救的核糖体的时间提供了有价值的线索。综上所述，这些研究表明，高效的RNase R-核糖体结合需要活跃的反式翻译，并表明RNase R在将tmRNA开放阅读框确立为替代mRNA模板后的早期阶段捕获新出现的无义mRNA。
